First report of Echinococcus granulosus sensu stricto (G1) in Nigeria, West Africa.

Echinococcus granulosus sensu stricto is regarded to have the highest zoonotic potential of all Echinococcus taxa. Globally, human infection due to this species constitutes over 88.44% of the total cystic echinococcosis (CE) burden. Here, we report a CE infection in a Nigerian camel caused by E. granulosus G1 genotype. To the best of our knowledge, this report is the first encounter of the G1 genotype in the West Africa sub-region where the G6 genotype is reportedly prevalent, suggesting that the epidemiology of this highly zoonotic group could have a wider host range and distribution in the sub-region, and emphasizes the need for further investigation into the genetic diversity of Echinococcus spp. in Nigeria and across the sub-region.

[Advances in research on echinococcus shiquicus tapeworm].

Echinococcosis is an age-old disease that causes serious damage to the animal husbandry and the human health perennially. As a newly discovered species of Echinococus, E. shiquicus has the potential public health significance and could be a potential parasitic zoonosis. In this review, its etiology, life cycle, epidemiology, detection and diagnoses, public health etc. are discussed or summarized. Also, a series of comparisons among E. granulosus, E. multilocularis and E. shiquicus are made.

Transcriptomic analysis of the larva Taenia multiceps.

Taenia multiceps is an adult worm affiliated to Taeniidae family, Platyhelminthes phylum. The larvae of the parasite (Coenurus cerebralis) parasitic in the brain and spinal cord in domestic and wild ruminants or humans can led to a fatal central nervous system (CNS) disease. The aims of the present study were to define the transcriptome profiles of the larvae of T. multiceps by RNA-Seq approach, and to generate large functional gene datasets that could be used to predict the key molecular pathways linked to this cestode. Our results generated a total of 39,094,890 clean reads that were assembled from the sequence data in 90,833 contigs. Briefly, 70,253 unigenes with a mean length of 1492bp were formed. Based on a sequence similarity search against the databases (NR, Swissport, GO, COG, KEGG) using BLASTX with an E-value cutoff of 10-5, 40,465 of unigenes were identified as coding sequences (CDS) and 3261 were scanned by ESTScan. The present study carried out the transcriptome of the larval stage of T. multiceps, which provides a solid foundation for further studies in molecular biology and biochemistry as well as identification of candidate genes used in diagnosis and vaccine development.

Proteomic analysis of differentially expressed proteins in the three developmental stages of Trichinella spiralis.

Trichinella spiralis, an intracellular parasitic nematode, can cause severe foodborne zoonosis, trichinellosis. The life cycle of T. spiralis consists of adult (Ad), muscle larvae (ML) and newborn larvae (NBL). The protein profiles in different developmental stages of the parasite remain unknown. In the present study, proteins from lysates of Ad, ML and NBL were identified by isobaric tags for relative and absolute quantitation (iTRAQ). A total of 4691 proteins were identified in all the developmental stages, of which 1067 proteins were differentially expressed. The number of up-regulated proteins in NBL was higher than that of the other two groups. The protein profiles from Ad, ML and NBL were compared in pairs. The identified proteins were involved in various functions of T. spiralis life cycle, including sexual maturity, metabolism, utilization of carbohydrates, lipids and nucleotides, and other crucial developmental processes that occur at distinct stages. Further investigation of the transcriptional levels of major sperm protein, serine protease, zinc finger protein, etc. from the different protein profiles using quantitative RT-PCR showed identical results to the iTRAQ analysis. The differentially expressed proteins that are involved in developmental regulation and host-parasite interactions should be further studied.

Cloning and characterization of thioredoxin peroxidases from Trichinella spiralis.

The intracellular parasitic nematode, Trichinella spiralis, can initiate a high level of oxidative stress, especially during rapid growth and generative propagation phases. Thioredoxin peroxidases (TPXs) protect helminths against oxidative stress, but none has been identified in T. spiralis. Here, 3 members of the TPX family were cloned from T. spiralis muscle larvae (ML). The lengths of TsTPX ORFs were 747bp, 588bp and 594bp, respectively, and the deduced proteins predicted to contain AhpC-TSA and 1-cys Prx_C domains. Interestingly, qRT-PCR data showed that TsTPX genes were expressed in all three developmental stages of T. spiralis. The TsTPX2 and TsTPX3 genes were up-regulated in day 3 adults (Ad3) compared with newborn larvae (NBL) and ML (P<0.05); expression levels of the TsTPX1 gene in ML were higher compared with Ad3 and NBL amounts (P<0.05). After prokaryotic expression, the reactivity of rTsTPX proteins was assessed by Western-blotting: only rTsTPX1 was specifically recognized by T. spiralis infection sera from pigs. Enzyme catalytic experiments showed that rTsTPX proteins could deoxidize H2O2 in the presence of DTT, with the catalytic ability increasing with protein concentration and time.

Cloning, identification, and bioinformatics analysis of a putative aquaporin TsAQP from Trichinella spiralis.

Vaccination as a preventative strategy against Trichinella spiralis infection is an ongoing effort, although no ideal vaccine candidates have been identified until now. Identification of more effective antigens that have a role in essential life stages of the parasite and that may be effective vaccine candidates is therefore of importance. In the present study, we identified a novel aquaporin gene (TsAQP) from T. spiralis, and the potential antigenicity of TsAQP was evaluated by epitope prediction. A total of 11 post-translational modification sites were predicted in the protein and fell into 4 categories: N-glycosylation; casein kinase II phosphorylation; protein kinase C phosphorylation; and N-myristoylation sites. TsAQP is a membrane intrinsic protein with high hydrophobicity; the main hydrophobic domains comprised up to 38.5% of the protein and were distributed at amino acid positions 21-43, 54-71, 83-91, 107-121, 163-174, 187-200, and 242-261. The protein consisted mainly of helices (39.58%) and loops (50%). The advanced structure of TsAQP was predicted using homology modeling, which showed that the protein was formed from 6 membrane-spanning domains connected by 5 loops. Based on these analyses, 6 potential B-cell epitopes and 4 potential T-cell epitopes were further predicted. These results suggest that TsAQP could be a promising antigen candidate for vaccination against T. spiralis.

Molecular characterization of enolase gene from Taenia multiceps.

Taenia multiceps is a cestode parasite with its larval stage, known as Coenurus cerebralis, mainly encysts in the central nervous system of sheep and other livestocks. Enolase is a key glycolytic enzyme and represents multifunction in most organisms. In the present study, a 1617bp full-length cDNA encoding enolase was cloned from T. multiceps and designated as TmENO. A putative encoded protein of 433 amino acid residues that exhibited high similarity to helminth parasites. The recombinant TmENO protein (rTmENO) showed the catalytic and plasminogen-binding characteristics after the TmENO was subcloned and expressed in the pET30a(+) vector. The TmENO gene was transcribed during the adult and larval stages and was also identified in both cyst fluid and as a component of the adult worms and the metacestode by western blot analysis. Taken together, our results will facilitate further structural characterization for TmENO and new potential control strategies for T. multiceps.

Detection of anti-Trichinella antibodies in serum of experimentally-infected swine by immunochromatographic strip.

An immunochromatographic strip method, developed with the excretory-secretory antigens from muscle larvae (ML) of Trichinella spiralis labeled with colloidal gold, was used for the detection of anti-Trichinella antibodies in serum of experimentally-infected swine. Sera from swine infected with 200, 2000 and 20,000 infective ML were collected at different days post infection (dpi) and used to evaluate the method. The strip method was shown able to detect anti-Trichinella antibodies by 35 dpi, 28 dpi and 21 dpi for the three different infection doses, respectively, and closely correlated with the results of an ELISA test. The strip method is rapid and easy to perform and is suggested as an acceptable alternative for clinical laboratories lacking specialized equipment, and for field diagnosis of trichinellosis.

Unique antigenic gene expression at different developmental stages of Trichinella pseudospiralis.

Parasite-induced and parasite-regulated larval capsule formation and host immunosuppression are two major characteristics that are unique in Trichinella spp. infections, but the molecule(s) and mechanism(s) that mediate these processes remain largely unknown. Trichinella pseudospiralis and Trichinella spiralis, are obviously different with respect to these two characteristics. A comparative study of these two species, in particular their antigen expression profiles at different developmental stages (the main molecules involved in the cross-talk or interaction between each parasite and its host), may help us better understand the parasite molecules and mechanisms involved. Here, we constructed cDNA libraries from T. pseudospiralis adults (Ad), newborn larvae (NBL) and muscle larvae (ML) mRNA and screened them with pig anti-T. pseudospiralis serum collected 26, 32 and 60 days post-infection (p.i.). The most abundant antigens were found to vary among life-cycle stages. Pyroglutamy peptidase 1-like and 6-phosphogluconolactonase-like genes predominated in the Ad stage and a serine protease (SS2-1-like gene) predominated in NBL similar to that observed in T. spiralis. Muscle larvae expressed proteasome activator complex subunit 3-like and 21 kDa excretory/secretory protein-like genes. This study indicated that parasites of two species may utilise different molecules and mechanisms for larvae capsule formation and host immunosuppression during their infections. Proteins of antigenic genes identified in this study may be also good candidates for diagnosis, treatment or vaccination for T. pseudospiralis infection, and also for the differential diagnosis of two species' infections.

Identification of antigenic genes in Trichinella spiralis by immunoscreening of cDNA libraries.

Genes encoding antigens of adult worm, newborn larvae and muscle larvae of Trichinella spiralis were identified by immunoscreening their corresponding cDNA libraries. The cDNA libraries of T. spiralis adult (3 day old, Ad3) and newborn larvae (NBL) were screened using the serum of a pig infected with 20,000 muscle larvae (ML) at 26 days post-infection (dpi). Fifty-two positive clones representing 18 unique genes were obtained from the Ad3 cDNA library. The deduced amino acid sequences of 8 cDNAs were sequence homologues of the serine protease-like protein family. In the screening of NBL cDNA library, 81 positive clones representing 8 unique genes were isolated and of these, 70 clones corresponded to an NBL stage-specific serine protease gene. The ML cDNA library was screened using pig anti-Trichinella serum obtained at 60 dpi and 18 positive clones representing 8 unique genes were identified. Ten clones encoded a protein that is identical to a T. spiralis serine protease inhibitor. In general, our results revealed that antigenic serine protease-like proteins were found during the two invasive stages, Ad and NBL when these libraries were screened using 26 dpi antiserum, whereas a serine protease inhibitor was found in abundance in the ML library when it is screened using 60 dpi antiserum. These data are consistent with serine proteases playing an important role during the invasive stages of Trichinella infections, but become inhibited or internally controlled when the parasite enters a more stable, non-developing environment.

Species identification of Trichinella isolates from China.

Two species of Trichinella were identified from China by means of PCR amplification of the mitochondrial small subunit ribosomal DNA and the expansion segment V region of the ribosomal DNA. Seven isolates originating from domestic pig and one isolate originating from dog showed identical DNA banding pattern to Trichinella spiralis, and two isolates from dog showed DNA banding pattern identical to Trichinella nativa. Sequence analysis of the 5S rDNA inter-gene spacer region from the ten Trichinella isolates confirmed the existence of only two Trichinella species and revealed the inner species genetic variation within T. spiralis and T. nativa.

Trichinella spiralis--a potential anti-tumor agent.

Murine forestomach carcinoma (cell line MFC), ascitic hepatoma (cell line H22) and sarcoma (cell line S180) solid tumor models were used to test the anti-tumor effect of Trichinella spiralis in vivo. Mice previously infected by oral administration of 400 viable T. spiralis larvae per mouse for 7 days were grafted with various solid tumor cell lines. Other groups of tumor-bearing mice were given caudal vein injection of crude extracts of adult and newborn larvae at 17.5, 35.0 or 70.0 mg kg(-1). These treatments to inhibit tumor growth were dose-dependent (p<0.05). The anti-proliferative activity of crude T. spiralis extract was examined in vitro at 0.035, 0.070 or 0.140 mg ml(-1) using MFC, H22, S180, human chronic myeloid leukemia cell line (K562) and hepatoma cell line (H7402), tumor cell proliferation in vitro was measured by methyl thiazolium stain and was inhibited in dose-dependent manner (p<0.05). At the same doses, crude T. spiralis extracts induced apoptosis of K562 and H7402 as detected by DNA fragmentation. Cell cycle analysis indicated that crude T. spiralis extracts, at 0.140 mg ml(-1), arrested the cell cycle of K562 and H7402 in G1 or S phase. It is concluded that T. spiralis contains anti-tumor active agent.

Identification of stage-specifically expressed genes of Trichinella spiralis by suppression subtractive hybridization.

SUMMARYNewborn larvae (NBL) and adult (Ad) stage-specifically expressed genes or members of gene families of Trichinella spiralis were identified by suppression subtractive hybridization (SSH). Six cDNA clones were identified as NBL stage-specific, including 1 member of the T. spiralis gene family encoding glutamic acid-rich proteins, 2 clones encoding novel serine proteases, 2 closely related clones encoding proteins that are members of a deoxyribonuclease II (DNase II)-like family and 1 clone with no similarity to known genes. Four stage-specific clones encoding homologues of retinoid X receptor, caveolin, C2H2 type zinc finger protein and a putative protein with no homology to known sequences were obtained from 3-day-old adult worms. One gene specifically up-regulated in the 5-day-old adult worms encoding a putative cuticle collagen was also identified.

Cloning and analysis of a novel cDNA from Trichinella spiralis encoding a protein with an FYVE zinc finger domain.

A cDNA library from Trichinella spiralis adults 3 days post-infection was screened with a cDNA probe, designated T 54, derived from a newborn larvae subtracted cDNA library. Sequence analysis showed that the positive clone contained a cDNA insert of 1464 bp in length with a single open reading frame of 1290 bp, which encoded a protein of 429 amino acids with a putative molecular mass of 49.9 k Da. Database analysis predicted the deduced protein had a leucine zipper motif and an FYVE zinc finger domain. The recombinant fusion protein was expressed and rabbit anti-recombinant protein sera reacted with a single peptide migrating at approximately 55 k Da in crude worm extract from muscle larvae, adults and newborn larvae stages.

A stage-specific open reading frame from three-day old adult worms of Trichinella spiralis encodes zinc-finger motifs.

The aim of the study was to isolate genes coding for stage-specific antigens of T. spiralis. Such antigens may then be associated with local and systemic immune responses against adult T. spiralis. Recombinant clones were obtained with an adult stage specific probe from a cDNA library of three-day old adult T. spiralis. Several cDNA clones encoding the same peptide were identified and their stage specificity was confirmed by northern blot analysis. Three independent clones were fully sequenced, and the resulting sequence found to code for a 487 amino acid peptide with a deduced molecular weight of approximately 55 kDa. Sequence analysis showed that the 55 kDa peptide contained putative DNA binding motifs, suggesting that this protein may be involved in transcriptional regulation during the early development of the parasite.

Molecular cloning of a cDNA encoding a putative cuticle collagen of Trichinella spiralis.

A 5-day-old adult stage-specific cDNA fragment from Trichinella spiralis was identified by suppression subtractive hybridization and was used as a probe to screen the cDNA library. The cDNA sequence coding for a putative T. spiralis cuticle collagen was isolated. The cDNA encoded an open reading frame of 343 amino acid residues with molecular weight of 35.1 k Da. The deduced protein contained an N-terminal signal peptide, a nematode cuticle collagen N-terminal domain and a collagen triple helix repeat domain. Searches in GenBank using BLASTP showed up to 47% identity to cuticle collagens from other nematodes. Southern blot analysis of genomic DNA indicated this gene was present as a single copy in T. spiralis genome.